1. Field of the Invention
The present invention relates to a novel peptide ligand i.a., to be used to impair cancer cell migration, in particular breast and colon cancer, and to act as a tumour suppressor in thyroid carcinoma and neurocarcinoma, In particular it relates to a hexapeptide ligand.
2. Brief Description of the Art
The Wnt proteins are a family of secreted glyco-proteins with a molecular weight of 39-46 kDa that participate in development and tumourigenesis via autocrine or paracrine routes (for review see (1-3)). Secreted Wnt proteins bind to and activate G-protein-coupled receptors of the Frizzled (Frz) family (4, 5). The LDL-receptor related proteins LRP5 or LRP6 are presumed to act as co-receptors in this context (6, 7). Based on the ability of different Wnt proteins to transform mouse mammary epithelial cells, thy can be divided into three different classes. Wnt-1, -3a, and -7a have the greatest transforming capacity; Wnt-2, -5b, and -7b have an intermediate transforming capacity; and Wnt-4, -5a, and -6 are non-transforming (8).
It has previously been reported that a low-level of Wnt-5a protein expression in a primary invasive breast carcinoma is associated with higher histological grade (poor differentiation) and a shortened recurrence-free survival due to a more rapid development of distant metastases (9). This association is not due to an effect of Wnt-5a on proliferation, since no correlation between loss of Wnt-5a protein expression and the proliferative marker Ki67 has been detected (9). The only presently available explanation for how the presence of Wnt-5a can reduce the metastatic capacity of invasive breast cancer is its ability to enable increased adhesion, presumably via the collagen-activated discoidin domain receptor 1 (DDR1), however it is possible that as yet undefined mechanisms could also contribute to the described clinical effect of Wnt-5a on tumour progression. Despite this, a Wnt-5a-dependent activation of DDR1 results in decreased motility and invasive potential of breast epithelial cancer cells (10). In breast epithelial cells that are normally firmly adhesive, a decreased expression of Wnt-5a and thus a decreased adhesion will increase their motility. In accordance, it has been observed that by stably introducing a Wnt-5a expression vector into a MCF-7 breast cancer cell line low in endogenous expression of Wnt-5a, was required for collagen-induced activation of the adhesion receptor DDR1 (10). In concordance, transfection of the normal human mammary epithelial cell line HB2 (11) stably transfected with Wnt-5a or antisense Wnt-5a caused a respective increased or decreased adhesion of these cells to collagen I (12). In human mammary cells it is not yet established via which of the different Frz-receptors that Wnt-5a mediates its effects. However, in both fibroblast-like synoviocytcs and in malignant melanoma cells it has been clearly shown with a specific blocking Ab that the receptor responsible for the Wnt-5a-induced effects is Frz-5 (13, 14).
To develop a specific treatment for breast cancer metastasis based on the functional role of Wnt-5a it is important to understand the cellular and molecular events responsible for its effects on this epithelial tumour. One of the critical and initial events in cancer metastasis is the ability of the transformed cells to de-adhere from each other and also from ECM components of the basement membrane (15). In the presence of Wnt-5a, the adhesion receptor DDR1 receptor can be activated by collagen in breast epithelial cells and is therefore a likely candidate that could mediate the Wnt-5a-induced increased adhesion of these cells to collagen (10). A possible explanation for the elevated metastatic activity seen in Wnt-5a lacking breast carcinomas could be a reduced adhesion and a subsequent increased motile activity In these tumour cells. One approach to reduce the metastatic activity in breast carcinomas lacking Wnt-5a protein expression could therefore be to find a way to overcome the translational defect responsible for the loss of Wnt-5a protein expression (16) and another to reconstitute the Wnt-5a-induced activation of the cells. Experimentally, addition of recombinant Wnt-5a to differentiated thyroid carcinomas has been shown to have tumour suppressor activity (17). However, addition of a large 43 kDa Wnt-5a protein to a patient, most likely over an extended period of time, does not emerge as a very attractive approach.
Previous work in platelets by Andersen and co-workers (18) has identified two different hexapeptide fragments that specifically can activate the G-protein-coupled protease-activated receptors PAR1 and PAR4, respectively. Other well-studied small peptides that can specifically bind and activate G-protein-coupled receptors on mammalian cells with high potency are the bacterial derived formylated peptides that bind to and activate the chemotactic peptide receptor on leukocytes (19). In this situation the formyl group has been shown to profoundly increase the binding affinities for the peptides to the receptor and consequently also for amplifying the activation of the cells (19). Interestingly enough, these peptides are functionally active even at a low pH such as that encountered at sites of inflammation. The pH at inflammatory sites is most likely lower than the estimated pH in a solid tumour (20, 21).